To address the problem of manure-based environmental pollution in the pork industry, we have developed the phytase transgenic pig. The saliva of these pigs contains the enzyme phytase, which allows the pigs to digest the phosphorus in phytate, the most abundant source of phosphorus in the pig diet. Without this enzyme, phytate phosphorus passes undigested into manure to become the single most important manure pollutant of pork production. We show here that salivary phytase provides essentially complete digestion of dietary phytate phosphorus, relieves the requirement for inorganic phosphate supplements, and reduces fecal phosphorus output by up to 75%. These pigs offer a unique biological approach to the management of phosphorus nutrition and environmental pollution in the pork industry.

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Bacteroides succinogenes S-85 grows readily in media containing 0.2% (w/v) filter paper cellulose or microcrystalline cellulose as the carbohydrate source. During growth, the cells appear to adhere to the cellulose. Cell-free culture supernates and cell extracts from cellulose-grown cultures had very low hydrolytic activity against either filter paper or crystalline cellulose (Avicel) as substrate, although H3PO4-swollen cellulose, carboxmethylcellulose, and cellobiose were readily hydrolyzed. Cells grown on either cellobiose or glucose exhibited cell-bound carboxymethylcellulase (CMCase) and cellobiase activities. Cultures grown on cellulose had seven to eight times more CMCase activity than either cellobiose- or glucose-grown cultures. Seventy percent of the CMCase activity was present in the supernate, of which 50--60% was associated with sedimentable membranous fragments. the cellobiase, which was largely cell associated, appeared to be constitutive, and the only product detected on enzymic hydrolysis of cellobiose was glucose. The cellobiase activity was strongly inhibited by 0.02 M tris(hydroxymethyl)-aminomethane (Tris), pH 7.1, but this was partially relieved by phosphate ions. These data indicate that B. succinogenes S-85 contains high endo-beta-1,4-glucanase and beta-1,4-glucanase and beta-1,4-glucosidase-like activities.

Two endoglucanases designated EG1 and EG2 were purified by column chromatography from the nonsedimentable extracellular culture fluid of Bacteroides succinogenes S85. They accounted for approximately 32 and 11%, respectively, of the total endoglucanase present in the nonsedimentable fraction. The most active enzyme (EG1) had a molecular weight of 65,000, pl of 4.8, and temperature and pH optima of 39°C and 6.4, respectively. The Km for carboxymethyl cellulose was 3.6 mg/ml, and the V.., was 84 U/mg. The major products of cellulose hydrolysis catalyzed by EG1 were cellotriose and cellobiose. EG2 was present as two components with molecular weights of 118,000 and 94,000. The two components had nearly identical cyanogen bromide peptide maps, thereby indicating that the 94,000-dalton component was a proteolytic degradation product of the 118,000-dalton enzyme. The larger component, which was more abundant in the culture fluid than the smaller form was, had a Km of 12.2 mg/ml and a V.x of 10.4 U/mg. It was a basic protein with a, pl of 9.4, a temperature optimum of 39°C, and a pH optimum of 5.8. The major product of cellulose hydrolysis was cellotetraose. EG2 exhibited specific binding to acid-swollen cellulose, whereas EG1 did not, and neither of them had affinity for crystalline cellulose. Based on the substrate specificities and the affinities of the two enzymes for cellulose, we postulated that EG2 is involved in the early stages of cellulose hydrolysis and that EG1 is active primarily on the products arising from EG2.Bacteroides succinogenes is a predominant cellulolytic bacterium in the bovine rumen (6,20). However, neither nongrowing cells nor cell-free culture fluid from B. succinogenes affects the extensive hydrolysis of crystalline cellulose (16). Therefore, our research was centered on determining the types of cellulolytic activities possessed by the bacterium, with the ultimate objective being to identify the enzymes responsible for the extensive hydrolysis of cellulose by growing cultures.During growth of B. succinogenes in continuous culture with cellulose as the carbon source, endoglucanase and chloride-stimulated cellobiosidase activities have been shown to be present in both the cells and the extracellular culture fluid (18). A cellodextrinase was detected in the periplasmic space, while cellobiase activity was membrane associated (18,19). Greater than 70% of the endoglucanase activity was present in the extracellular culture fluid of cells grown in either a chemostat culture or a batch culture after all of the cellulose was digested (11,16,19). Results of the batch culture study revealed that 50 to 62% of the extracellular endoglucanase was associated with sedimentable membrane fragments, 9 to 13% was associated with nonsedimentable material with a molecular weight greater than 4 x 106, and 28 to 38% was associated with molecules with a molecular weight of approximately 45,000, as determined by exclusion chromatography (15,16,31). The endoglucanase activities in these various fractions were further ...

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na+, K+, and Mg++ at their concentrations in the growth medium, when suspended briefly in 0.5 M sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 M NaCI. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 M sucrose. Since this material was released only when the outer doubletrack structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.

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Microbial inocula from rumen fluid, soil, and contents of the large intestines of chickens (CLIC) and of swine (SLIC) were tested for their ability to transform deoxynivalenol (vomitoxin) in vitro. Microorganisms in (CLIC) completely transformed pure vomitoxin, and this activity was retained through six serial subcultures. No alteration of the toxin by incubation with SLIC was detected, whereas 35% of the vomitoxin was metabolized in the original culture of rumen fluid and 50%o was metabolized by the soil sample, though metabolism was decreased in subsequent subcultures of either sample. A single metabolite was isolated and identified as deepoxy vomitoxin. The increase in concentration of deepoxy vomitoxin in the culture medium corresponded with the decrease in vomitoxin concentration. The vomitoxin transformation rate was not affected by either the ratio of CLIC to vomitoxin (5 to 0.2 g of CLIC per mg of vomitoxin) or the initial concentration of vomitoxin (14 to 1,400 ppm) in the medium. Biotransformation of vomitoxin was completely inhibited when the pH in the medium was lowered to 5.20. Sodium azide at a 0.1% (wt/vol) concentration in the medium blocked the transformation of vomitoxin, suggesting that the deepoxidation of vomitoxin is an energy-dependent process. About 50%o of the vomitoxin in moldy corn in culture medium was transformed by microorganisms from CLIC. The vomitoxin transformation rate in moldy corn was not affected when the concentration of CLIC changed from 0.2 to 0.8 g/ml of medium. Vomitoxin in the moldy corn was not transformed when CLIC were added to corn without culture medium. An acid pH due to accumulation of fermentation products, such as organic acids, may be a major factor which inhibits the complete transformation of vomitoxin in moldy corn treated with CLIC.